Human cytomegalovirus (CMV) is a ubiquitous agent in human populations. Infections are generally asymptomatic, but there can be serious medical manifestations of the disease in immunocompromised individuals (transplant recipients and AIDS patients) and in congenitally infected newborns. In immunodeficient patients, primary CMV infection and reactivation of latent virus is associated with serious disease including retinitis and pneumonia. CMV infection also predisposes the patient to fungal and bacterial infections. Congenital CMV infection of the fetus occurs in about 1% (36,000) of infants born in the U.S. per year. Of these infants 10-20% will have symptomatic infection at birth or within two years of birth with a mortality rate of 10-15%. Among the survivors, many will have mild to severe neurologic complications including hearing loss, learning disabilities and mental retardation.
Vaccines that prevent or reduce CMV-associated disease are clearly needed. The CMV (Towne) strain has been tested as a vaccine candidate in normal individuals and renal transplant patients (Quinnan, Jr., G. V. et al. (1984) Am Intern Med 101:478-483); (Plotkin, S. A. 1985, CMV Vaccines, In: The Herpes Viruses vol. 4, ed., Roizman and Lopez, Plenum Press, N.Y., p. 297-312). While this vaccine appeared to have no deleterious effects and did reduce symptoms of CMV disease in transplant recipients, there are many objections to the use of experimental live attenuated virus vaccines, including the possibility of immune impairment resulting from virus infection and reports of possible association between CMV and oncogenesis.
In the absence of a complete understanding of the biology of CMV, the most rational approach to a vaccine would involve the development of subunit vaccines based upon the surface glycoproteins of the virus using recombinant viral glycoproteins which elicit neutralizing antibodies.
Like other herpesviruses, CMV specifies multiple glycoproteins (Stinski, M. (1976) J Virol 19:594-609; Pereira, L., et al. (1982) Infect Immun 36:933-942). Characterization of these have involved studies of CMV-infected cells and purified virions using polyclonal and monoclonal antibodies (Pereira, L., et al. (1984) Virology 139:73-86; Britt, W. J. (1984) Virology 135:369-378; Nowak, B., et al. (1984) Virology 132:325-338; Law, K.M., et al. (1985) J Med Virol 17:255-266; Rasmussen, L., et al. (1984) Proc Natl Acad Sci USA 81:876-880; and Britt and Auger (1986) J Virol 58:185-191).
U.S. Pat. No. 4,689,225, issued Aug. 25, 1987 and based upon the work described in the Pereira et al. references, supra, describes a method and vaccine for CMV infections using a polypeptide designated therein as glycoprotein A (gA1-A7) of cytomegalovirus. Two glycoproteins designated p130 (gp130) and p55 (gp55) (based on the molecular weights given in kilodaltons) have been partially purified and shown to elicit a neutralizing response in guinea pigs (Rasmussen, L., et al. (1985) J Virology 55:274-280). The gp130 glycoprotein appears to be a precursor to the gp55 glycoprotein.
The gB gene from CMV strain AD169 (which appears to be similar to the pl30 CMV protein described by Rasmussen et al., supra) has been identified by nucleotide sequencing (Cranage, M. P. et al. (1986) EMBO J 5(11):3057-3063) with a 906 amino acid protein deduced therefrom. The gB gene product was expressed in recombinant vaccinia virus and rabbits immunized with this gene product produced antibodies that immunoprecipitate gB from CMV-infected cells and neutralize CMV infectivity in vitro (See also WO 87/05326).
Although there is much ongoing activity towards both the identification of major gylcoproteins which are the targets for viral neutralization and the development of a subunit CMV vaccine, to date, the origin of the gp55 CMV glycoprotein has not been established nor has gp55 been identified by nucleotide or amino acid sequence and therefore, no vaccine composed of the 55,000 dalton recombinant viral gB protein or any truncated recombinant polypeptide thereof has been reported. Clearly, in light of the absence of a complete understanding of the biology of CMV, it would be desirable to provide a safe, effective and economic vaccine capable of affording protection against cytomegalovirus infections, as well as to provide diagnostic reagents capable of detecting the particular immunogenic stimulus resulting from CMV infections.
Disclosure of the Invention
The present invention provides recombinant polypeptides derived from the 55,000 dalton protein derived from gB and truncated fragments thereof which contain an epitope which is immunologically identifiable with one encoded by the CMV genome. A recombinant polypeptide derived from the gp55 CMV glycoprotein gB is provided in one embodiment of the invention.
The complete characterization of the gp55 protein derived from gB permits the production of polypeptides which are useful as standards or reagents in diagnostic tests and/or as components of vaccines. Since the desired polypeptide can be synthetically made in a relatively pure form or by recombinant DNA technology, the problems with other methods of immunogen and vaccine manufacture, including coproduction of competitive antigens and contaminants, are avoided.
In a preferred embodiment of the invention, the truncated gp55 gB fragment contains an epitope that is immunologically reactive with a CMV neutralizing antibody. The neutralizing antibody can be generated by techniques known in the art such as that described for monoclonal antibodies disclosed in Rasmussen et al., supra and U.S. Pat. No. 4,689,225.
Also provided in another preferred embodiment of the invention is a recombinant polypeptide encoded within CMV glycoprotein gB, which has a modified endoproteolytic cleavage site such that cleavage of the gB protein is effectively inhibited. The modification of the cleavage site is accomplished using site specific mutagenesis on the DNA encoding the polypeptide at or near the proteolytic cleavage site. Related to this aspect of the invention are the polynucleotides encoding the recombinant polypeptides.
Another aspect of the invention is a recombinant gp55 polynucleotide comprising a nucleotide sequence derived from the CMV gB gene. Related to this aspect of the invention are truncated recombinant polynucleotides containing regions encompassing nucleotides 1381 through 2040 of gp55 and nucleotides 1381 through 1938 of gp55, which regions contain an epitope which is immunologically reactive with a CMV neutralizing antibody.
Yet another aspect of the invention provides an expression system comprising host cells transformed with a vector containing the recombinant polynucleotides of the invention.
Another aspect of the invention provides a vaccine or prophylactic agent against human cytomegalovirus infection, said vaccine comprising a recombinant gp55 polypeptide derived from the CMV gB genome or the endoproteolytic cleavage site modified gB polypeptide in amounts effective to elicit viral neutralizing activity against cytomegalovirus when administered to a susceptible individual.
Still another aspect of the invention provides a vaccine against human cytomegalovirus infection, said vaccine comprising a recombinant polypeptide derived from a truncated fragment encoded within CMV glycoprotein gB wherein the truncated fragment contains an epitope which is immunologically reactive with a CMV neutralizing antibody, said recombinant polypeptide being present in an immunologically acceptable carrier in an amount effective to elicit viral neutralizing activity against cytomegalovirus when administered to a susceptible individual.
Another aspect of the invention provides for a DNA hybridization assay for detecting CMV homologous sequences in a biological sample comprising: a) incubating a biological sample with a DNA probe, which probe may be optionally labeled with an enzyme, radioactive tag or a fluorescent tag, under conditions which promote the formation of DNA duplexes, wherein said DNA probe is derived from gp55 nucleotide sequences; and b) detecting the formed DNA duplexes containing the DNA probe.
A further aspect of the invention provides an immunoassay for detecting antibodies directed against a CMV antigen in a biological specimen comprising:
(a) incubating a biological sample with a probe polypeptide under conditions which allow the formation of an antibody-antigen complex, wherein said probe polypeptide consists of the p55 CMV recombinant protein or a truncated fragment thereof and said protein or truncated fragment contains an epitope which is immunologically reactive with a CMV-neutralizing antibody; and
(b) detecting an antibody-antigen complex containing the probe antigen.
Yet another aspect of the invention provides polyclonal antibodies against the recombinant gp55 or truncated polypeptides thereof, for immune prophylaxis.
Other and further aspects of the present invention will be apparent from the following description and claims and other embodiments of the invention employing the same or equivalent principles may be used by those skilled in the art without departing from the present invention and the purview of the appended claims.